FUNCTIONAL ANALYSIS OF CFTR

CFTR的功能分析

• 批准号: 6239178
• 负责人: PETER C MALONEY
• 金额: $ 12.76万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-29 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6239178
• 关键词: Xenopus oocyte; chloride channels; cysteine; cystic fibrosis; gene mutation; intermolecular interaction; molecular biology; molecular cloning; molecular pathology; protein structure function; serine; site directed mutagenesis; transfection

The goal of this study is to understand structure/function relationships in CFTR, the protein whose defect causes cystic fibrosis (CF). To do this, we will study the hydrophobic sector of this complex molecule, because this sector of CFTR is most closely associated with the Cl channel function absent in CF. Two broad questions will be asked. One set of experiments will identify elements important to Cl channel activity by implanting cysteine residues to serve as receptors for cysteine-specific reagents. The Cl channel function of such variants will be evaluated in Xenopus oocytes. Among variants that retain function, we will examine more closely those in which channel activity is irreversibly blocked by hydrophilic cysteine-modifying agents. Analysis of a pool of such candidate mutants should identify the subset of transmembrane segments surrounding the CFTR Cl channel and the specific residues lining the channel pore. To complement these studies, parallel experiments will use mutagenesis to identify residues that may interact as salt bridges in CFTR. The genesis of such work relates to conflicting reports regarding the role of two charged residues (E92 and K95) in the first transmembrane segment of CFTR. We believe the present data are resolved if these two interact with one another in a direct way. To test the hypothesis that an ion pair is functionally relevant in this and other areas, we will increase charge density by replacing the anionic partner with a cationic residue (and vice versa) and examine the phenotype of the variant CFTRs in oocytes. To determine whether the original residues interact with one another, we will then replace them, one at a time, with neutral amino acids. In these latter trials, direct evidence for a salt bridge will arise if function is found only when oppositely charged or neutral residues are present. Together, such experiments should add significantly to our understanding of the membrane domain of CFTR, leading to a better grasp of how the CFTR Cl channel functions and is regulated.

本研究的目的是了解结构/功能之间的关系 在CFTR中，其缺陷导致囊性纤维化(CF)的蛋白质。去做 这个，我们将研究这个复杂分子的疏水部分， 因为CFTR的这一部分与CL关系最密切 CF中缺少通道功能。我们将提出两个广泛的问题。一 一系列实验将确定对氯离子通道重要的元素 通过植入半胱氨酸残基作为受体的活性 半胱氨酸特异性试剂。这些变体的氯离子通道功能 将在非洲爪哇的卵母细胞中进行评估。在保留了 函数，我们将更仔细地检查其中的通道活动 被亲水性半胱氨酸修饰剂不可逆转地阻断。 对这些候选突变体库的分析应该识别出子集 围绕CFTRCl通道的跨膜片段和 特定的残留物排列在通道孔内。 为了补充这些研究，平行实验将使用突变。 鉴定在CFTR中可能作为盐桥相互作用的残基。这个 这类工作的起源与关于角色的相互矛盾的报道有关 第一跨膜段的两个带电残基(E92和K95) Cftr.我们相信，如果这两者相互作用，目前的数据就会得到解决 以一种直接的方式相互联系。来检验一个离子的假设 Pair在这一领域和其他领域具有功能相关性，我们将增加 用阳离子残基取代阴离子伙伴的电荷密度 (反之亦然)，并检查变异CFRR的表型 卵母细胞。以确定原始残基是否与 另一种，然后我们将用中性氨基取代它们，一次一个 酸。在后面的审判中，盐桥的直接证据将 如果仅当相反电荷或中性时才找到函数，则出现 有残留物存在。 总之，这样的实验应该会大大增加我们的理解 ，从而更好地掌握CFTR是如何 CL通道起作用并受到调节。